In a normal pregnancy, the fertilized egg implants itself within the uterine endometrial lining. With an ectopic pregnancy, the fertilized egg is implanted outside of the uterine endometrial lining. Depending upon the actual site of implantation, an ectopic pregnancy may be classified, for example, as a tubal pregnancy, a cervical pregnancy, an ovarian pregnancy, or a peritoneal pregnancy. More than 95% of ectopic pregnancies are tubal pregnancies, which therefore is often used as a general designation for an ectopic pregnancy. The etiology of ectopic pregnancy includes previous tubal ligation (contraception), PID (Pelvic Inflammatory Disease), administration of ovulation controlling formulations and STD (Sexually Transmitted Disease). The incidence of ectopic pregnancy is quite high, approaching as many as one per about 64-241 pregnant females.
Ectopic pregnancy is one of the most frequent obstetric and gynecologic emergencies, and its incidence increases year by year. Observable symptoms of tubal rupture due to an ectopic pregnancy, may include lower abdominal pain, amenorrhea, vaginal bleeding, dizziness due to a blood pressure drop, as well as, nausea and vomiting due to the gastrointestinal symptoms. Untreated tubal rupture may lead to intraperitoneal bleeding, which has emerged as the most significant cause of early stage death of pregnant females. In other words, if an ectopic pregnancy is not discovered at an early stage, tubal rupture and the associated shock from bleeding may lead to death of the patient.
Unfortunately, due to its lack of specific symptoms, and the lack of any effective method of determining the presence of an ectopic pregnancy, diagnosis of this potentially fatal condition is difficult. An efficient method of detecting ectopic pregnancy in its early stages could greatly reduce maternity-related mortality rates, and significantly reduce the anxiety associated with the uncertainty that the pregnancy is normal. Recognizing the value and importance of an efficient and effective early stage diagnostic method for determining ectopic pregnancy, the present inventors conducted extensive studies, and have successfully discovered an efficient diagnostic device for distinguishing between a normal pregnancy and an ectopic pregnancy.
hCG is produced in syncytiotrophoblast of the placenta and induces a constant production of progesterone in the early stage of pregnancy, functioning to maintain the implantation until the tenth week of pregnancy, when the placenta has become completely functional. Other know functions of hCG include, but are not limited to, the stimulation of the maternal thyroid, the stimulation of production of testosterone in the fetal testis, and plays an important role in the differentiation of internal genitalia in a male embryo. hCG is detected in the blood and urine of a pregnant female at a level of about 25 mIU/ml approximately 8 days after conception (fertilization). The concentration of hCG rises rapidly at a constant rate from the fifth week of pregnancy, whereby it reaches a concentration of about 1 IU/ml at the sixth week of pregnancy, and about 100-120 IU/ml at the tenth week of pregnancy. From this point forward, the hCG concentration decreases to reach about 20 IU/ml at the twentieth week of pregnancy, where it remains constant throughout the remainder of the pregnancy.
hCG is a glycoprotein comprised of two subunits, α and β, consisting of about 30% carbohydrates, and has a molecular weight of about 36,700 daltons. The α-subunit is comprised of 92 amino acid residues, having the same structure as the α-subunit of luteinizing hormone and thyroid stimulating hormone. The β-subunit is comprised of 145 amino acid residues (structurally characteristic of hCG) (Lapthom et al., Nature, 369:455-61, 1994).
Typically, hCG exists in various forms in human body fluids. Examples of forms of hCG include, for example, intact-hCG (I-hCG) and modified hCGs such as nicked hCG(N-hCG), hyperglycosylated hCG, free β-hCG and free β-core fragment. The various forms of hCG are produced during pregnancy, as well as, in the event of pituitary gland or trophoblast disease or choriocarcinoma. Intact hCG comprises about 90% of the total hCG present in a woman during a normal pregnancy, while various modified forms of hCG, such as nicked hCG and free β-hCG, comprise about 10% or less.
With an ectopic pregnancy, intact hCG comprises close to 100% of the total hCG concentration, and there are extremely small amounts of modified hCGs. In addition, the total hCG secretion, and its subsequent range of increase are significantly smaller as compared to that occurring in a female with a normal pregnancy. For example, the level of hCG secretion observed with an ectopic pregnancy is only about ⅕ of that observed with a normal pregnancy and, at the eighth week, is only about 1/50 of that seen with a normal pregnancy (i.e., about 15 IU/ml).
U.S. Pat. No. 5,786,220 discloses a process for the preparation of a one-step diagnostic reagent system that distinguishes between a normal pregnancy and an abnormal pregnancy. This reference discloses that normal pregnancy, spontaneous abortion, ectopic pregnancy, cancers, etc. can be diagnosed by simultaneously determining the concentrations of progesterone and hCG in the woman's bodily fluid. The patent teaches a method of diagnosis wherein a progesterone concentration of 25 ng/ml or lower in blood, together with an hCG concentration of about 25-2,500 mIU/ml is indicative of a spontaneous abortion or an ectopic pregnancy, and a progesterone concentration exceeding 25 ng/ml, together with an hCG concentration exceeding 2,500 mIU/ml is indicative of a normal intrauterine pregnancy.
Denil et al, Fertility & Sterility, 72:1013-17(1999) disclosed that the freeβ-hCG level in a normal pregnancy is typically 30-170 IU/ml; 1-70 IU/ml in an abnormal intrauterine pregnancy; and 0.059-29 IU/ml in an ectopic pregnancy, indicating that hCG degradation products are secreted to a much lower concentration in the ectopic pregnancy than in the normal pregnancy.
As discussed previously, hCG is the most important hormone in the diagnosis of pregnancy, and its level in the early stages of pregnancy, may be utilized in combination with ultrasound findings, to distinguish between a normal pregnancy, an abortion, and an ectopic pregnancy. When diagnosing pregnancy, the level of hCG in the woman's bodily fluid may be used at an early stage to confirm pregnancy. A low level of hCG, together with a rapid doubling of the hCG blood level every 1.4-2 days during the fifth to ninth weeks from conception, is typically indicative of either an abortion or an ectopic pregnancy. A greatly elevated hCG is interpreted as being indicative of multiple pregnancy or hydatidiform mole.
Ectopic pregnancy may also be diagnosed with the use of ultrasonography, diagnostic laparoscopy and the like in addition to the measurement of serum and urine hCG level in a pregnant female. Particularly, ultrasonography is conducted for diagnosis in combination with the hCG level measurement, wherein the presence of gestational sac in the ultrasound findings together with an hCG level below 1,000 mIU/ml indicates a sparse viability of the pregnancy. If the hCG level does not rise quantitatively by at least 65% per 48 hour, the prognosis of the pregnancy is determined to be very poor.
Unfortunately, the known methods of determining ectopic pregnancy, such as measuring the doubling time of the hCG level and the measuring the free β-hCG level in the body fluid of a pregnant female, are time consuming and require repeated blood sampling. The method described in the U.S. Pat. No. '220 patent is based on diagnostic principles different than those of the present invention in that the '220 method requires the contemporaneous measurement of both progesterone and hCG concentrations. Additionally, even though the distinction between a normal pregnancy and an abnormal pregnancy can be made, an effective early distinction between an ordinary spontaneous abortion and a potentially fatal ectopic pregnancy is very difficult to determine.
Accordingly, the present inventors have conducted extensive studies on the process for the preparation of a one-step simultaneous diagnosis kit by which the diagnosis of a normal pregnancy or an ectopic pregnancy can be achieved at a very early stage. As a result, the present inventors have provided a one-step diagnostic kit by which a normal pregnancy can be determined by detecting an hCG level of 25 mIU/ml, and an ectopic pregnancy can be determined through comparison between the concentrations of I-hCG and modified hCGs.